Hollow protein nano-particles and method of introducing substances into cells

ABSTRACT

As a versatile means for specifically and safely transporting and transferring substances (genes, proteins, compounds, etc.) into target cells and tissues, hollow nano particles comprising a protein capable of forming particles (e.g., hepatitis B virus surface antigen protein), and a biorecognition molecule introduced therein, are provided.

This application is a 371 of PCT/JP01/00926 filed Feb. 9, 2001.

TECHNICAL FIELD

The present invention relates to hollow nano particles, which comprise aprotein capable of forming particles, to which a biorecognition moleculehas been introduced. More particularly, the invention relates to hollownano particles, which can be used as transporters for transferringsubstances into particular cells or tissues.

BACKGROUND ART

In recent years, in the field of medicine, active development of highlyeffective drugs, which act directly on the affected part with less sideeffect, is being carried out. Particularly, a method known as drugdelivery system (DDS) has attracted a great deal of attention since itallows the specific transportation of effective components such as drugsto target cells or tissues, and enables the effective component to actat the target site.

Further, in the field of molecular cell biology, gene transfer to aparticular cell is recognized as an essential technology and is beingstudied actively. Additionally, with the recent discoveries in thegenetic background of various diseases by the progress of the HumanGenomic Project, the realization of such highly specific methods forgene transfer would also enable application in the field of genetherapy.

As for the method for transferring a gene into cells, a method ofincorporating a macromolecular form of gene by endocytosis (calciumphosphate method, lipotectamine method, etc.), and a method forinserting a gene into a cell by perforating the cell membrane usingelectric pulse stimulation (electroporation or gene gun method) areknown, and being used generally in molecular biology experiments.

Although these methods are convenient, they may not readily be appliedin vivo, because the site to which genes are introduced must be exposedsurgically by the direct and physical wounding of the cells. Inaddition, it is difficult to attain a gene transfer efficiency close to100%.

Alternatively, as a highly safe method of transferring a substance, theliposome method is known. This method can be applied to cells andtissues of a living body because it does not require the injuring of thecells. However, it is difficult to give high cell and tissue specificityto liposome, which is a simple lipid. Moreover, there is another problemthat the gene transfer efficiency in vivo is much lower than therequired value.

Recently, a new technique of gene transfer, which uses an infectiousvirus created by integrating the gene of interest into the virus DNA,has been developed. This technique can be applied to individuals withapproximately 100% of the gene transfer efficiency, does not require thegene transfer site to be exposed, and thus, has attracted much attentionas an epoch-making method: however, there is a serious problem that thegene is transferred to cells other than the target, because the virusinfects a wide range of cells non-specifically. Moreover, there is apossibility that the virus genome itself is integrated into thechromosome to induce unexpected side reaction in the future. Therefore,the technique has not yet been used therapeutically in early stages of adisease, and at present, has been applied only to patients of terminalsymptoms.

In view of the above situation, the present invention was made to solvethe problems of the prior art. Accordingly, the purpose of the presentinvention is to provide a versatile method for the safe and specifictransportation and transfer of a substance (gene, protein, compound, andthe like) to a target cell or tissue.

DISCLOSURE OF THE INVENTION

As a means to solve the above-described problems, the present inventionfirstly provides a hollow nano particle, comprising a protein capable offorming a particle, and a biorecognition molecule introduced thereto.

Secondly, the present invention provides a hollow nano particle,comprising a protein particle obtained by expressing a protein in aeucaryotic cell, and a biorecognition molecule introduced thereto.

As further embodiments, the present invention thirdly provides thehollow nano particle of the second invention, wherein the eucaryoticcell is either yeast or genetically recombinant yeast, and fourthly, thehollow nano particle, wherein the eucaryotic cell is an insect cell.

Further, the present invention fifthly provides the hollow nanoparticle, wherein the protein capable of forming a particle is ahepatitis B virus surface antigen protein.

Furthermore, the present invention sixthly provides the hollow nanoparticle, wherein the hepatitis B virus surface antigen protein is oneof which its antigenicity has been reduced.

The present invention also provides as additional embodiments, any ofthe above-described hollow nano particles, wherein seventhly, thebiorecognition molecule is a cell function-regulating molecule,eighthly, an antigen, ninthly, an antibody, and tenthly, a sugar chain.

Eleventhly, the present invention further provides a transporter ofsubstances, comprising any of the above-described hollow nano particles,and a substance that is to be introduced into cells incorporatedtherein.

The present invention also provides as additional embodiments, thetransporter of substances, wherein twelfthly, the substance to beintroduced is a gene, thirteenthly, a protein, fourteenthly, an RNasethat shows cytotoxicity in the cell, and fifteenthly, a compound.

The present invention further provides a method of preparing thetransporter of substances of any one of the eleventh to fifteenthinvention, comprising sixteenthly, the insertion of the substance to thehollow nano particle of any one of the first to tenth invention byelectroporation, seventeenthly, by ultrasonication, eighteenthly, bysimple diffusion, and nineteenthly, by using a charged lipid.

The invention also provides twentiethly, a method for transferring asubstance into cells or tissues, which comprises the use of the hollownano particles of any one of the first to tenth invention, ortwenty-firstly, a method for transferring a substance into cells ortissues, which comprises the use of the transporter of substances of anyone of the eleventh to fifteenth invention.

Moreover, twenty-secondly, the present invention provides a therapeuticmethod for treating diseases, which comprises transporting a substanceto certain cells or tissues by using at least one of the above twentiethor twenty-first method of transferring a substance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram, which shows each protein region of an HBsAg gene inthe Examples of the present invention. 1 to 8 indicates the function ofthe respective moiety of the surface, antigen (1: release inhibition; 2:receptor; 3: sugar chain 1; 4: polymerized serum albumin receptor; 5:transmembrane; 6: stabilization; 7: sugar chain 2; 8: transmembrane lowpolymerization/secretion).

FIG. 2 is a diagram, which shows the expression and purificationprocedures for HBsAg particles using recombinant yeast in the Example ofthe present invention. Each represents: (a) Preparation of recombinantyeast; (b) Culture on a High-Pi medium; (c) Culture on an 8S5N-P400medium; (d) Homogenization; (e) Density gradient centrifugation; and (f)HBsAg particles.

FIG. 3 shows a confocal laser fluorescent micrograph of HepG2, whenHepG2 is in contact with HBsAg particles incorporating a GFP plasmid inthe Example of the present invention. Each represents: (a) using HBsAgparticles (plasmid: 8 ng); (b) Using lipid (liposome) (plasmid: 800 ng);(c) vector only (plasmid: 800 ng).

FIG. 4 shows a confocal laser fluorescent micrograph, indicating thatGFP is also expressed in cells derived from human hepatogenic carcinoma(NUE) by HBsAg particles incorporating GFP plasmid. Each represents: (a)HepG2 cell and (b) NUE cell.

FIG. 5 shows a confocal laser fluorescent micrograph, indicating thatHBsAg particles allow highly specific introduction of GFP into cellsderived from human hepatic carcinoma (HuH-7). Each represents: (a)tumorigenic area of mice (positive fluorescence) to which cells derivedfrom human hepatogenic carcinoma was transplanted; (b) normal mice liver(no fluorescence).

BEST MODE FOR CARRYING OUT THE INVENTION

The hollow nano particle of the present invention enables the specifictransportation of a substance to a target cell or tissue by transferringa biorecognition molecule into a protein capable of forming particles.As a protein capable of forming particles, subviral particles obtainedfrom a variety of viruses may be applied. Specifically, hepatitis Bvirus (HBV) surface antigen protein is exemplified.

The protein particle comprising a protein capable of forming a particle,include those obtained by expressing the protein in a eucaryotic cell.That is, when a protein capable of forming a particle is expressed in aeucaryotic cell, the protein is expressed and accumulated as a membraneprotein on the membrane of the endoplasmic reticulum membrane and isreleased as particles. As the eucaryotic cell, yeast or recombinantyeast, and insect cells are applicable.

The present inventors have reported, as described in the followingExamples, that when the L protein of the above-described HBV-surfaceantigen is expressed in the recombinant yeast, elliptical hollowparticles of about 20 nm in diameter and about 150 nm in length, withlarge amounts of the protein embedded within the lipid bilayer membranesderived from yeast, were found from the expressed HBV-surface antigen Lprotein (J. Bio. Chem. Vol. 267, No. 3, 1953-1961, 1992). Since theseparticles contain no HBV genome or other HBV proteins, they do not actas viruses and are highly safe to the human body. In addition, theparticles are effective as transporters for the specific transportationof substances to liver cells, because they display on their surface ahepatocyte-specific receptor that bear the high infectivity of HBV toliver cells.

Thus, the method of forming protein particles using recombinant yeast ispreferred, because the particles are produced from soluble proteins incell lysates at high efficiency.

On the other hand, insect cells are eucaryotic cells closer to higheranimals than yeast, and are preferable for the efficient production ofvarious proteins, because they can reproduce higher order structuressuch as sugar chains, which yeast cannot reproduce. Conventional systemusing insect cells utilized baculoviruses, and protein expression wasaccompanied by the expression of the virus, which led to cell death andlysis. Consequently, there was a problem that the continuous expressionof the protein was required, and that the protein was degraded byprotease released from the dead cells. Additionally, it was difficult topurify the protein expressed in a culture medium, because a large amountof fetal bovine serum existed in the medium. Recently, however, aninsect cell system without the use of baculovirus, which may be culturedunder serum-free conditions, was developed and marketed by invitrogen.Accordingly, by using such insect cells, it is possible to obtainprotein particles that are purified easily, and in which high-orderstructure is reproduced.

In the protein hollow nano particles of the present invention, byconverting the receptor on the surface of the particles obtained by thevarious methods described above into an optional recognition molecule,or by transferring a variety of substances (DNAs, RNAs, proteins,peptides, and drugs) into the particles, the transportation ofsubstances to cells and tissues other than liver cells at a considerablyhigh specificity becomes possible.

Of course, the proteins capable of forming particles are not limited tothe above-described hepatitis B virus surface antigen and may be anykind of proteins as long as they are able to form particles; naturallyoccurring proteins derived from animal cells, plant cells, viruses,microorganisms, and the like, as well as various synthetic proteins areapplicable. Further, for example, if there is a possibility that anantigenic protein of viral origin induces the generation of antibodiesin the body, it may be used as a biorecognition molecule aftermodification to reduce its antigenicity.

As the biorecognition molecules that is introduced into a proteincapable of forming particles, for example, cell function-regulatingmolecules, that is, molecules that regulates cell function such asgrowth factor, cytokines, etc.; cell or tissue-recognizing moleculessuch as cell surface antigen, tissue specific antigen, receptor, etc.;molecules derived from viruses or microorganisms; antibodies, sugarchains, lipids, and the like may preferably be used. These maybe chosenproperly according to the target cells or tissues.

According to the present invention, a substance desired to be introducedinto target cells or tissues (substance to be introduced into cells) isincorporated in the protein hollow nano particles as described above, toform a transporter of a substance that shows cell specificity. Thesubstance to be introduced into cells, which is incorporated in thetransporter, includes, for example, genes such as DNAs and RNAs, naturalor synthetic proteins, oligonucleotides, peptides, drugs, natural orsynthetic compounds, and the like.

Specifically, human RNase I (Jinno H., Ueda M., Ozawa S., Ikeda T.,Enomoto K., Psarras K., Kitajima M., Yamada H., Seno M., Life Sci.,1996, 58 (21), 1901-8), and RNase 3 (also known as ECP: EosinophilCationic Protein; Mallorqui-Fernandez G., Pous J., Peracaula R., AymamiJ., Maeda T., Tada H., Yamada H., Seno M., de Llorens R., Gomis-Ruth FX, Coll M., J. Mol. Biol., Jul. 28, 2000, 300 (5), 1297-307), which hasbeen reported by the present inventors are applicable.

It Is known that these proteins show no cytotoxic activity outside thecell, but are cytotoxic when incorporated in the cells. Therefore, byusing the transporters of the present invention incorporating RNase,proteins that exhibit selective cytotoxic activity against cells andtissues can be expressed forcibly, and is expected to be a newtherapeutic method for cancer treatment.

Further, as a method of transferring such substances into theabove-described hollow nano particles, various methods generally used inchemical or molecular-biological experimental techniques are applicable.For example, electroporation, ultrasonication, simple diffusion, or useof a charged lipid is preferably exemplified.

Hence, by using such protein hollow nano particles or transporters ofsubstances, the in vivo or in vitro introduction of substances intocells or tissues are enabled. In addition, as exemplified above forRNase, the use of the protein hollow nano particles or transporterspermits the introduction of a substance into certain cells or tissuesfor treatment of various diseases or as one step of such treatment.

Hereinafter, embodiments for carrying out the invention are described infurther detail by the following examples and the attached figures. Ofcourse, the invention is not limited in any way by the followingexamples. It is needless to say that various modifications of theembodiment are allowed.

EXAMPLES

In the following Examples, HBsAg indicates a hepatitis B virus surfaceantigen. HBsAg, which is a coat protein of HBV, is classified into threetypes of protein, i.e., S protein, M protein and L protein, as indicatedin FIG. 1. The S protein is an important coat protein common to allthree proteins. M protein contains an added 55 amino acid residues(pre-S2 peptide) at the N-terminus of the S protein. Further, L proteincontains 108 or 119 amino acid residues (pre-S1 peptide) added to theN-terminus of the M-protein.

The Pre-S regions of the HBsAg L protein (pre-S1, pre-S2) are both knownto play an important role in the binding of HBV to the liver cells:Pre-S1 has a direct binding site for the liver cells, and pre-S2has apolymerized album in receptor which binds to the liver cells mediated bypolymeric albumin in blood.

When HBsAg is expressed in eucaryotic cells, the HBsAg proteins areexpressed and accumulated as membrane proteins on the endoplasmicreticulum membrane. L protein of HBsAg is aggregated, and released asparticles into the lumen side in a budding form, while incorporating theendoplasmic reticulum membrane.

In the following Examples, L protein of HBsAg was used. FIG. 2illustrates the expression and purification procedure for HBsAgparticles as described in the following Examples.

Example A Expression of HBsAg Particles by the Recombinant Yeast

The recombinant yeast carrying pGLDLIIP39-RcT (Saccharomyces cerevisiaeAH22R strain) was cultivated in a synthetic medium High-Pi and 8S5N-P400to express HBsAg L-protein particles, based on the method reported bythe present inventors in J. Bio. Chem. Vol. 267, No. 3, 1953-1961 (1992)(FIGS. 2 a and b).

From the recombinant yeast in the stationary growth phase (after about72 hours), whole cell extracts were prepared using Yeast ProteinExtraction Reagent (Pierce Chemical Co.), then separated by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), andsubjected to silver staining to identify HBsAg in the sample.

HBsAg was found to be a protein with a molecular weight of about 52 kDa.

Example B Purification of HBsAg Particles from the Recombinant Yeast

(1) The recombinant yeast (wet weight: 26 g) cultivated on the syntheticmedium 8S5N-P400 was suspended in 100 ml of Buffer A (7.5 M urea, 0.1 Msodium phosphate, pH 7.2, 15 mM EDTA, 2 mM PMSF, 0.1% Tween 80) andhomogenized by a BEAD-BEATER using glass beads. Then, the supernatantwas recovered by centrifugation (FIGS. 2 c and d).

(2) Subsequently, the supernatant was mixed with 0.75-fold volume of 33%(w/w) PEG 6000, and cooled over ice for 30 minutes. Then, the mixturewas centrifuged (7000 rpm, 30 minutes) to recover pellets. The pelletswere then re-suspended in Buffer A without Tween 80.

(3) The re-suspended solution was layered over CsCl solution of 10-40%gradient and subjected to ultracentrifugation at 28000 rpm for 16 hours.After centrifugation, the sediment was divided into 12 fractions, whichwere subjected to Western Blotting (primary antibody was anti-HBsAgmonoclonal antibody) to identify the fraction containing HBsAg. Further,the fraction containing HBsAg was dialyzed in Buffer A without Tween 80.

(4) The dialysate (12 ml) obtained in (3) was layered over a sucrosesolution of 5-50% gradient and subjected to ultracentrifugation at 28000rpm for 16 hours. In the same manner as in (3), the fraction containingHBsAg after centrifugation was identified and dialyzed in Buffer Acontaining 0.85% NaCl instead of urea and Tween 80. ((2)-(4): FIG. 2 e)

(5) The procedure of (4) was repeated and the sample after dialysis wasconcentrated using Ultra Filter Q2000 (Advantech Co.) and refrigeratedat 4° C. until use. (FIG. 2 f)

From the result of Western Blotting (3) after CsCl density equilibriumcentrifugation, HBsAg was found to be a protein with a molecular weightof 52 kDa and an S-antigenicity. A total of about 24 mg of purifiedHBsAg particles were obtained from 26 g (wet weight) of the yeast cellsderived from 2.5 L of culture medium.

The fractions obtained in the course of purification were analyzed bysilver-staining SDS-PAGE. Further, to confirm that protease derived fromyeast was removed by purification, the HBsAg particles obtained in (5)were incubated at 37° C. for 12 hours, then subjected to SDS-PAGE, andidentified by silver staining.

As a result, it was confirmed that protease of yeast origin wascompletely removed by the overall purification process.

Example 1 Gene Transfer by HBsAg Particles in Human Liver Cancer CellHepG2

Human liver cancer cell HepG2 in the logarithmic growth phase wasinoculated on a 3.5 cm glass-bottom petri dish at 1×10⁵ cells/well andcultivated overnight in a D-MEM medium containing 10% fetal bovine serumunder 5% CO₂ at 37° C. On the following day, the HBsAg particles weremixed with green fluorescence protein expression plasmid (GFP expressionplasmid pTB701-hGFP) and subjected to electroporation, then added to aHepG2 culture medium and cultivated in 5% CO₂ at 37° C. for 4 days.

The state of expression of GFP in HepG2 was observed with a confocallaser fluorescent microscope.

The gene transfer efficiency of HBsAg particles was determined by usingHepG2. HBsAg particles containing GFP plasmid were prepared by anelectroporation at a condition of 110V and 950 μF using a cuvette of 4mm gap, and used to transform HepG2 cells, followed by cultivation inD-MEM under 5% CO₂ at 37° C. for 4 days.

FIG. 3 shows the confocal laser micrographs of HepG2. By comparing FIG.3( a) with FIGS. 3( b) and (c), the efficiency shown in FIG. 3( a) wasapproximately 200 times that of FIG. 3( b), indicating that the transferefficiency of GFP expression plasmid by HBsAg particles was very high.

Example 2 Gene Transfer by HBsAg Particles in Human Liver Cancer CellsOther than HepG2

According to the method as described in Example 1, HuH-7 (JCRB0403) andNUE (offered by Professor Takuji Tadakuma, Parasitology Lab, NationalDefense Medical College) were prepared as human hepatic cancer cells.

Further, as negative controls, human colon cancer cells, WiDr (ATCCCCL-218), HT29 (ATCC HTB-38) and SW1116 (ATCC CCL-233), human malignantmelanoma cells SEKI (JCRB0620), and human squamous cell carcinoma A431(JCRB9009) were cultivated separately on 3.5 cm glass bottom petridishes, of which 10⁵ cells were infected by HBsAg particles containingthe GFP expression plasmid (pEGFP-F (Clontech)) and cultivatedcontinuously for 4 days. Then, the expression of GFP in the cells wasobserved with a confocal laser fluorescent microscope.

As a result, the degree of fluorescence in the Huh-7 and NUE cells wasobserved to be approximately equal to that of HepG2 cells (FIG. 4).

On the other hand, no GFP fluorescence was observed in the cells notderived from human liver.

Accordingly, it was demonstrated that, using the HBsAg particles of theinvention, a gene could be introduced into human liver cells at thecultured cell level, in high efficiency and specificity.

Example 3 Gene Transfer by HBsAg Particles into Nude Mice with HumanLiver Cancer Transplanted Thereto

Tumor-bearing mice were prepared by subcutaneously injecting 1×10⁷ cellsof the human tumor strains (HuH-7 and WiDr) used in Example 2 to thebilateral dorsal cutis of nude mice (line: BALB/c nu/nu; microbiologicalquality: SPF; sex: male, 5 weeks old), and growing them for 2 to 6 weeksuntil the transplanted tumor developed into solid cancer of about 2 cmin diameter.

Ten μg of the HBsAg particles containing 2.5 μg of GFP expressionplasmid (pEGFP-F) obtained according to the method described in Example2 were administered intraperitoneally to mice with a 26G needle. Fourdays after administration, the mice were sacrificed, and the tumorportion, liver, spleen, kidney and intestine were removed, and theirtissues were fixed and embedded using a resin-embedding kit for GFP(Technovit 7100).

Specifically, the fixation was performed by immersion in 4% neutralizedformaldehyde and dehydration was carried out in 70% EtOH at roomtemperature for 2 hours, in 96% EtOH at room temperature for 2 hours,and in 100% EtOH at room temperature for 1 hour. Pre-immersion wascarried out in an equal amount mixture of 100% EtOH and Technovit 7100at room temperature for 2 hours. Then, the tissues were immersed inTechnovit 7100 at room temperature within a period of 24 hours, takenout, and allowed to stand at room temperature and then at 37° C. for 1hour for polymerization.

Sections were prepared according to conventional methods, and stainedwith hematoxin-eosin (conventional method of tissue staining); thefluorescence of GFP for the HBsAg particle-administered group and thenon-administered group were compared under a fluorescent microscope(FIG. 5).

As shown in FIG. 5, fluorescence generated by GFP was observed on thetumor portions of the mice bearing cancer induced by HuH-7 cells derivedfrom human liver cancer. However, no fluorescence was observed in theliver, spleen, kidney, and intestine simultaneously removed from themice. Moreover, no fluorescence was seen in the tumor potion, liver,spleen, kidney, and intestine of mice bearing cancer induced by WiDrcells derived from human colon cancer, or in the control group to whichno HBsAg particles were administered.

Accordingly, it was demonstrated that, using the HBsAg particles of theinvention, a gene could be introduced into human liver cells in highefficiency and specificity, even in experimental animals. Therefore, thetransporter of substances of the present invention was confirmed to bevery effective.

Example C Preparation of a Multi-purpose Type of HBsAg Particles(HBsAg-Null Particles) for Displaying a Biorecognition Molecule

HBsAg particles are able to infect specifically to human liver cells,and the hepatocyte-recognizing site, which exhibit high infectivity,displayed on the surface of the particles is reported to be found in the3rd to 77th amino acid residues of the pre-S1 region (Le Seyec J.,Chouteau P., Cannie I., Guguen-Guillouzo C., Gripon P., J. Virol. 1999,March: 73(3), 2052-7).

Here, a method for preparing modified HBsAg particles (hereinafterreferred to as HBsAg-Null particles), which lacks its high infectivityto liver cells and can display an optional biorecognition molecule onits surface while maintaining the capability of forming particles.

In the plasmid pGLDLIIP39-RcT described in Example A, in order toeliminate the genetic region coding the human liver cell-recognitionsite and to introduce the restriction enzyme NotI site (gcggccgc) (SEOID NO: 23) at the same time, PCR was carried out with QuickChange™Site-Directed Mutagenesis Kit (Stratagene Co.) for the plasmidpGLDLIIP39-RcT, using the oligonucleotide of SEQ ID NO:1 and theoligonucleotide of SEQ ID NO:2 as PCR primers.

Specifically, using Pfu DNA polymerase (Stratagene Co.) as thermostableDNA polymerase, PCR was carried out under the schedule of: denaturationat 95° C. for 30 seconds; annealing at 55° C. for 1 minute; andpolymerization reaction at 68° C. for 30 minutes, 30 cycles. Then, thePCR product was treated with restriction enzyme DpnI, and transformedinto Esaherichia coli DH5α, after which the vector DNA was extractedfrom the resulting colony, and the mutated pGLDLIIP39-RcT plasmid(hereinafter, referred to as pGLDLIIP39-RcT (null)) was screened basedon its nucleotide sequence.

According to the method described in Example A, the plasmidpGLDLIIP39-RcT (null) was transformed and cultivated in synthetic mediumHigh-Pi and in 8S5N-P400, to express HBsAg-Null particles.

From recombinant yeast in the stationary growth phase (about 72 hoursafter starting the incubation), call extracts were prepared using anYeast Protein Extraction Reagent (product of Pierce Chemical Co.), andthen separated by SDS-PAGE, after which HBsAg-Null was identified bysilver staining and Western blotting using an anti-S monoclonal antibody(Funakoshi).

Thus, HBsAg-Null was found to be a protein with a molecular weight ofabout 42 kDa.

In addition, according to the method described in Example B, about 3 mgof purified HBsAg-Null particles were obtained from the above-describedcells (about 26 g) derived from 2.5 L of the culture medium. Measurementof S-antigenicity of HBsAg particles and HBsAg-Null particles (thedegree of particle-formation) using the Auszyme II EIA kit (Dainabot Co.Ltd.) gave about equal values for both proteins.

Example 4 Gene Transfer with HBsAg-Null Particles in Cancer Cells ofHuman Origin

According to the method described in Example 2, the GFP expressionplasmid (pEGFP-F (Clontech Co.)) was incorporated into the HBsAg-Nullparticles obtained in Example C, which lack the high infectivity to theliver cells, and are able to display an optional biorecognitionmolecule; gene transfer to HepG2 cells and a variety of cancer cells ofhuman origin as described in Example 2 was performed for cultured cells.

However, no fluorescence by GFP was observed in any of the cells. Thisindicates that the high infectivity to cells is absent in HBsAg-Nullparticles.

Example 5 Gene Transfer by HBsAg-Null Particles into Nude Mice withHuman Cancer Cells Transplanted Thereto

According to the method described in Example 3, mice bearing cancer wereprepared by transplanting of human tumor strains (HuH-7 and WiDr), andthe HBsAg-Null particles containing GFP expression plasmid (pEGFP-F(Clontech Co.)) were administered. However, no fluorescence by GFP wasobserved in the tumor portions or in any of the major organs.

From these results, it was confirmed that the HBsAg-Null particles haveno infectivity to any of the organs.

Example D Preparation of HBsAg Particles Displaying an Epidermal GrowthFactor (EGF)(HBsAg-EGF Particles)

The EGF receptor is known to be expressed on the surface of variouscells, and to be particularly associated with the deterioration ofcertain cancers (esophageal cancer, colon cancer, etc.). Hence, HBsAgparticles targeting the EGF receptor may provide an effective means forthe treatment of cancer tissues that express the EGF receptor.

Herein, a method for preparing HBsAg particles of EGF-displaying type(HBsAg-EGF particles) based on the HBsAg-Null particles obtained by themethod of Example C is described.

Using a cDNA fragment of EGF precursor of human origin (Bell G I, Fong NM, Stempien M M, Wormsted M A, Caput D, Ku L L, Urdea M S, Rall L B,Sanchez-Pescador R, Nucleic Acids Res. 1986, November, 11:14 (21),8427-46) as a template, a gene fragment coding a mature human EGF region(53 amino acid residues) was amplified by PCR according to conventionalmethods.

The two PCR primers used were the oligonucleotide of SEQ ID NO.3 forsense and the oligonucleotide of SEQ ID NO.4 for antisense, which wereboth designed to contain the restriction enzyme NotI site (gcggccgc)(SEQ ID NO: 23) at the 5′-terminus.

After separation of the PCR product by agarose electrophoresis, the bandcontaining the intended cDNA band (about 170 bp) was recovered, andsub-cloned to pCR2.1-TOPO vector (Invitrogen Co.) using TOPO TA Cloningkit (Invitrogen Co.). After confirming the nucleotide sequence, this wascleaved with the restriction enzyme NotI to recover the intended DNAfragment of about 170 bp, and using a TaKaRa Ligation kit ver.2 (TaKaRaCo.), cyclized together with pGLDLIIP39-RcT (null), which was firstcleaved with the restriction enzyme NotI, after which it was transformedinto Escherichia coli DH5α.

After screening by nucleotide sequence analysis, the fused plasmid inwhich the reading frame of the inserted EGF gene was identical to thatof the HBsAg gene was selected and designated as pGLDLIIP39-RcT-EGF.

According to the method of Example A, the plasmid pGLDLIIP39-RcT-EGF wastransformed and cultivated in the synthetic medium High-Pi and 8S5N-P400to express HBsAg-EGF particles.

From the recombinant yeast in the stationary growth phase (72 hoursafter starting incubation), crude cell extracts were prepared usingYeast Protein Extraction Reagent (Pierce Chemical Co.), separated bySDS-PAGE, and subjected to silver staining and Western blotting using ananti-human EGF polyclonal antibody (Santa Cruz Co.) to identify theHBsAg-EGF.

Thus, HBsAg-EGF was found to be a protein with a molecular weight ofabout 50 kDa.

According to the method described in Example B, about 3 mg of thepurified HBsAg-EGF particles were obtained from the above-describedcells (about 26 g) derived from 2.5 L of the culture medium. TheS-antiganicity (the degree of particle-formation) of HBsAg particles andHBsAg-EGF particles was measured using the Auszyme II EIA kit (DainabotCo. Ltd.), which can only detect the particle structure of HBsAg. Thesame values were observed for both.

Therefore, it was demonstrated that HBsAg-EGF particles were obtained,as were the HBsAg particles.

Example 6 Gene Transfer with HBsAg-EGF Particles in Cancer Cells ofHuman Origin

According to the method described in Example 2, the GFP expressionplasmid pEGFP-F vector (Clontech Co.) was incorporated into theHBsAg-EGF particles, and gene transfer to HepG2cells as well as avariety of human cancer cells described in Example 2 was performed forcultured cells.

Among them, strong fluorescence from GFP was observed in A431 cells,which expressed a large amount of EGF receptor on the surface.

Therefore, it was confirmed that the HBsAg-EGF particles possessedhigher infectivity to cells that express EGF receptor. Further, it wasdemonstrated that a biorecognition function could be given arbitrarilyto modified HBsAg particles at the cultured cell-level.

Example 7 Gene Transfer by HBsAg-EGF Particles into Nude Mice with HumanCancer Cells Transplanted Thereto

Cancer-bearing mice transplanted with human tumor strains (A431, HuH-7,WiDr) were prepared according to the method described in Example 3, andthe HBsAg-EGF particles containing a GFP expression plasmid (pEGFP-F(Clontech Co.)) was administered to them; strong fluorescence wasobserved at the tumor area from A431. On the other hand, no fluorescencefrom GFP was observed in the tumor areas from the other cells or in themajor organs.

Accordingly, it was confirmed that the HBsAg-EGF particles are able tospecifically infect cells that express considerable amounts of EGFreceptor, and that the particles have no infectivity to major organs.

Therefore, it was demonstrated that the HBsAg-Null particles with anoptional biorecognition molecule fused or added to them, are capable ofspecifically transporting substances to any desired tissue or organ.

Example E Preparation of HBsAg Particles (HBsAg-BTC Particles)Displaying Betacellulin (BTC)

BTC belongs to the type of EGF family, but its expression site isdifferent from that of EGF. Particularly, it has been found that BTCplays an important role in the differentiation of the β-cells of spleenLangerhans islet, which plays an important role in the blood sugarregulation mechanism. Accordingly, if HBsAg particles that target theBTC receptor could be prepared, they may be expected to be useful as aneffective means for transporting substances to tissues expressing theBTC receptor for the treatment of diabetes mellitus caused by β-cells.

Here, a method for preparing HBsAg particles of the BTC-displaying type(HBsAg-BTC particles) based on the HBsAg-Null particles obtained by themethod of Example C, is described.

Using a cDNA fragment of BTC precursor of human origin (Sasada R, Ono Y,Taniyama Y. Shing Y, Folkman J, Igarashi K; Biochem. Biophys. Res.Commun. Feb. 15, 1993: 190 (3), 1173-9) as a template, a gene fragmentcoding for a known site capable of binding to the BTC receptor (48 aminoacid residues of GHSFR - - - VDLFY) was amplified by PCR according toconventional methods.

The two PCR primers used were the oligonucleotide of SEQ ID NO:5 forsense and the oligonucleotide of SEQ ID NO:6 for anti-sense, which wereboth designed to contain the restriction enzyme NotI site (gcggccgc)(SEQ ID NO: 23) at the 5′-terminus.

After separation of the PCR product by agarose electrophoresis, the bandcontaining the intended cDNA band (about 160 bp) was recovered, andsub-cloned to cCR2.1-TOPO vector (Invitrogen Co.) using TOPO TA Cloningkit (Invitrogen Co.). After confirming its nucleotide sequence, this wascleaved with the restriction enzyme NotI to recover the intended DNAfragment of about 160 bp, and using a TaKaRa Ligation kit ver.2 (TaKaRaCo.), cyclized together with pGLDLIIP39-RcT (null), which was firstcleaved with the restriction enzyme NotI, after which it was transformedinto Escherichia coli DR5α. After screening by nucleotide sequenceanalysis, a fused plasmid in which the inserted BTC gene was identicalto the HBsAg gene in the reading frame was screened and designated aspGLDLIIP39-RcT-BTC.

According to the method of Example A, the plasmid pGLDLIIP39-RcT-BTC wastransformed and cultivated in a synthetic medium High-Pi and 8S5N-P400to express HBsAg-BTC particles.

From the recombinant yeast in its stationary growth phase (72 hoursafter starting incubation), crude cell extracts were prepared usingYeast Protein Extraction Reagent (Pierce Chemical Co.), separated bySDS-PAGE, and subjected to silver staining and Western blotting using ananti-human BTC polyclonal antibody (prepared by Mr. Seno, Department ofTechnology, Okayama University) to identify the HBsAg-BTC.

Thus, HBsAg-BCT was found to be a protein with a molecular weight ofabout 50 kDa.

According to the method described in Example B, about 3 mg of thepurified HBsAg-BTC particles were obtained from the above-describedcells (about 26 g) derived from 2.5 L of the culture medium. TheS-antigenicity of HBsAg particles and HBsAg-BTC particles (the degree ofparticle formation) was measured using Auszyme II EIA kit (Dainabot Co.Ltd.), which can only detect the particle structure of HBsAg. The samevalues were observed for both particles.

Example 8 Gene Transfer with HBsAg-BTC Particles in Cancer Cells ofHuman Origin

According to the method described in Example 2, a GFP expression plasmid(pEGFP-F (Clontech Co.)) was incorporated into the HBsAg-BTC particles,and gene transfer to rat pancreas cell AR42J expressing BTC receptor,human lung cancer cell H69 expressing no BTC receptor, as well as thevarious cancer cells of human origin described in Example 2 wasperformed at the cultured cell-level.

Among them, strong fluorescence by GFP was observed in AR42J cells whichexpressed a large amount of BTC receptor on the surface.

Thus, it was confirmed that the HBsAg-BTC particles have highinfectivity against the BTC receptor expression cells.

Example F Preparation of HBsAg Particles Displaying Basic FibroblastGrowth Factor (bFGF) (HBsAg-bFGF Particles)

In the growth of cancer tissues in individuals, it is known that asignal is sent by the cancer cells to the peripheral tissues to inducegeneration of blood vessels (angiogenesis). It is known that a varietyof growth factors (also known as angiogenesis factors) are involved,among which bFGF plays the central role. It has also been reported thatthe bFGF receptor is enhanced in the peripheral tissues (Li V W,Folkerth R D, Watanabe H, Yu C, Rupnick M, Barnes P, Scott R M, Black PM, Sallan S E, Folkman J, Lancet 1994, July 9:344 (8915), 82-6).

Therefore, it is considered that if HBsAg particles targeting the bFGFreceptor are prepared, they may be an effective means for transportingsubstances to tissues expressing the bFGF receptor, and therapy foreffectively inducing inhibition of angiogenesis in the peripheraltissues of cancers.

Herein, a method for preparing HBsAg particles of bFGF-displaying type(HBsAg-bFGF particles) based on the HBsAg-Null particles obtained by themethod of Example C, is described.

Using a cDNA fragment of bFGF precursor of human origin (Kurokawa T,Sasada R, Iwane M, Igarashi K, FEBS Lett. 1987, March 9:213 (1) 189-94)as a template, a gene fragment coding a known site capable of binding tothe bFGF receptor (146 amino acid residues of PALPED - - - PMSAKS) wasamplified by PCR according to conventional methods.

The two PCR primers used were the oligonucleotide of SEQ ID NO:7 forsense and the oligonucleotide of SEQ ID NO:8 for anti-sense, which wereboth designed to contain the restriction enzyme NotI site (gcggccgc)(SEQ ID NO: 23) at the 5′-terminus.

After separation of the PCR product by agarose electrophoresis, the bandcontaining the intended cDNA band (about 450 bp) was recovered, andsub-cloned to cCR2.1-TOPO vector (Invitrogen Co.) using TOPO TA Cloningkit (Invitrogen Co.). After confirming its nucleotide sequence, this wascleaved with the restriction enzyme NotI to recover the intended DNAfragment of about 450 bp, and using a TaKaRa Ligation kit ver.2 (TaKaRaCo.), cyclized together with pGLDLIIP39-RcT (null), which was firstcleaved with the restriction enzyme NotI, after which it was transformedinto Escherichia coli DH5α. After screening by nucleotide sequenceanalysis, the fused plasmid in which the inserted bFGF gene wasidentical to the HBsAg gene in the reading frame was screened anddesignated as pGLDLIIP39-RcT-bFGF.

According to the method of Example A, the plasmid pGLDLIIP39-RcT-bFGFwas transformed and cultivated in a synthetic medium High-Pi and in8S5N-P400 to express HBsAg-bFGF particles.

From the recombinant yeast in its stationary growth phase (72 hoursafter starting incubation), crude cell extracts were prepared usingYeast Protein Extraction Reagent (Pierce Chemical Co.), separated bySDS-PAGE, and subjected to silver staining and Western blotting using ananti-bFGF monoclonal antibody 3H3 (Wako Pure Chemical Ind.) to identifythe HBsAg-bFGF. Thus, HBsAg-bFGF was found to be a protein with amolecular weight of about 58 kDa.

According to the method described in Example B, about 2 mg of thepurified HBsAg-bFGF particles were obtained from the above-describedcells (about 26 g) derived from 2.5 L of the culture medium. TheS-antigenicity of HBsAg particles and HBsAg-pFGF particles (the degreeof particle formation) was measured using Auszyme II EIA kit (DainabotCo. Ltd.), which can only detect the particle structure of HBsAg. Thesame values were observed for both particles.

Example 9 Gene Transfer with HBsAg-bFGF Particles in Cancer Cells ofHuman Origin

According to the method described in Example 2, a GFP expression plasmid(pEGFP-F (Clontech Co.)) was incorporated into the HBsAg-bFGF particles,and gene transfer to human breast cancer cell MDA-MB-231 expressing bFGFreceptor, human squamous cell carcinoma A431 expressing no bFGFreceptor, as well as the various cancer cells of human origin describedin Example 2 was performed at the cultured cell-level.

Among them, strong fluorescence by GFP was observed in MDA-MB-231 cells,which expressed a large amount of bFGF receptor on the surface.

Thus, it was confirmed that the HBsAg-bFGF particles have highinfectivity against the bFGF receptor expression cells.

Example G Construction of Human RNase Expression Vector Incorporated inthe HBsAg Particles

Here, a vector that can express the above-described RNase in cells wasconstructed. First, using human RNase 1 gene (Seno, M., Futami, J.,Kosaka, M., Seno, S. and Yamada, H., Biochim. Biophys. Acta 1218 (3),466-468, 1994) as a template, a gene fragment coding human RNase 1 thatcontains a signal peptide (RO fragment) (156 amino acid residues) wasamplified by PCR, using the oligonucleotides of SEQ ID NO:9 (containingXhoI site, ctcgag (SEQ ID NO: 24)) and SEQ ID NO:10 (containing HindIIIsite, aagctt (SEQ ID NO: 25)) as primers.

Next, using the human RNase 1 gene as a template, a gene fragment codinghuman RNase 1 that contains no signal peptide(RM fragment) (128 aminoacid residues) was amplified by PCR, using the oligonucleotides of SEQID NO:11 (containing a XhoI site, ctcgag (SEQ ID NO: 24)) and SEQ IDNO:12 (containing a HindIII site, aagctt (SEQ ID NO: 25)) as primers.

In addition, using human ECP gene (Rosengerg H F, Ackerman S J and TenenD G, J. Exp. Med 170 (1), 163-176, 1989) as a template, a gene fragmentcoding human ECP that contains a signal peptide (EO fragment) (160 aminoacid residues) was amplified by PCR, using the oligonucleotides of SEQID NO:13 (containing a XhoI site, ctcgag (SEQ ID NO: 24)) and SEQ IDNO:14 (containing a HindIII site, aagctt (SEQ ID NO: 25)) as primers.

Further, by using the human ECP gene as a template, a gene fragmentcoding human ECP that contains no signal peptide (EM fragment) (133amino acid residues) was amplified by PCR, using the oligonucleotides ofSEQ ID NO:15 (containing a XhoI site, ctcgag (SEQ ID NO: 24)) and SEQ IDNO:16 (containing a HindIII site, aagctt (SEQ ID NO: 25)) as primers.

The resulting fragments RO, RM, EO and EM were then sub-cloned intopGEM-Teasy vector (Promega Co.), and after confirming the nucleotidesequences, the fragments were cleaved with EcoRI and HindIII andsubjected to agarose electrophoresis to recover the DNA fragmentscontaining the above fragments. On the other hand, the expression vectorpTriEx-1 (Novagen Co.) was cleaved with EcoRI and HindIII and cyclizedwith each of the above fragments using TaKaRa Ligation kit ver.2 (TaKaRaCo.). The resulting plasmids were designated as pTriEx-1-RO,pTriEx-1-RM, pTriEx-1-EO and pTriEx-1-EM.

Example H Cytotoxic Effect with the RNase Expression Vector in CulturedCells

COS-7 cells derived from the kidney of African Green Monkey wereinoculated in each well of a 16-well plate at 1×10⁴ cells/well, andcultivated overnight in D-MEM containing 10% fetal bovine serum under 5%CO₂ at 37° C. The following day, the plasmids, pTriEx-1-RO, pTriEx-1-RM,pTriEx-1-EO and pTriEx-1-EM were distributed at 0, 0.2, 0.5, 1.0, and5.0 μg each, and vigorously mixed with 3 μl of gene transfer lipidFuGene 6 (Roche), followed by the addition of 100 μl of serum-free D-MEMmedium to each well.

The mixture was cultivated for two nights in D-MEM containing 10% fetalbovine serum under 5% CO₂ at 37° C. Then, 100 μl each of MTT solution[PBS (phosphate-saline buffer) containing 5 mg/ml MTT (Wako PureChemical)] was added to each well, and allowed to stand at 37° C. for 4hours, followed by the addition of 1 ml of a solubilizer [0.04 Nhydrochloric acid in isopropanol]. After shaking at room temperature for1 hour, the absorption spectrum of the mixture was measured at 570 nmand 630 nm.

Each sample was divided into 3 parts, and the measured values wereobtained by dividing the absorbance at 570 nm by that at 630 nm. Theresults are shown in Table 1.

TABLE 1 Survival Rate (%) DNA RO RM EO EM Amount With No With No (μg)signal signal signal signal 0 100.0 100.0 100.0 100.0 0.2 87.9 87.8 97.493.3 0.5 81.3 77.7 80.8 94.9 1 77.3 84.9 86.0 89.0 5 69.2 79.7 70.0 96.7

Consequently, cytotoxicity-inducing ability was observed in all of theexpression systems.

From these results, it may be expected that by incorporating these RNaseexpression vectors into the various protein hollow nano particles of thepresent invention, the above-described RNase may be introducedcell-specifically and exhibit cytotoxic effects in the cells, therebyproviding an effective therapy for diseases.

Example I Preparation of HBsAg Particles with Serum-free Cultured InsectCells

Although the method of expressing HBsAg particles by recombinant yeastas described in Example A is a highly efficient method for preparing theHBsAg particles, in which approximately 40% of soluble proteins in thecells are converted into HBsAg, this method requires complicatedoperations such as those described in Example B, in order to obtainpurified HBsAg particles. In addition, although yeast has a proteinsynthesis system such as the endoplasmic reticulum membrane that issuitable for the expression of proteins derived from higher animals, itis known that, because it is a lower eucaryotic organism, it cannotreproduce higher order structure such as sugar chain.

Hence, hereinafter, a method for preparing HBsAg particles by an insectcell system, which does not require baculovirus and enables serum-freeculture, is described.

From the HBsAg expression plasmid pGLDLII39-RcT for yeast described inExample A, a chicken-derived lysozyme secretion signal peptidefusion-HBsAg gene was amplified by PCR, using the oligonucleotides ofSEQ ID NO:17 (containing a KpnI site, ggtacc (SEQ ID NO: 26) and SEQ IDNO:18 (containing a SacII site, ccgcgg (SEQ ID NO: 27) as primers.

After separating the PCR products by agarose electrophoresis andrecovering the intended cDNA band of about 1.3 kbp, it was sub-clonedinto pCR2.1-TOPO (Invitrogen Co.) using TOPO TA Cloning kit (InvitrogenCo.) and transformed into Escherichia coli DH5α. The plasmid, in whichthe intended gene was integrated correctly, was screened by nucleotidesequence analysis, treated with KpnI and SacII, and separated by agaroseelectrophoresis to recover the KpnK-SacII fragment of about 1.3 kbp.

Subsequently, the above gene fragment was ligated and cyclized betweenthe KpnI site and SacII site of the vector pIZT/V5-His (Invitrogen Co.)for stable expression of insect cells, using TaKaRa Ligation kit ver.2(TaKaRa Co.).

After confirming its nucleotide sequence, the plasmid was designated aspIZT/V5-His-HBsAg. Similarly, a modified HBsAg gene was removed from theplasmid pGLDLIIP39-Rct (null) described in Example C andpGLDLIIP39-RcT-EGF described in Example D, inserted into pIZT/V5-His,and designated as pIZT/V5-His-null and pIZT/V5-His-EGF, respectively.

On the other hand, the insect cell High Five strain (BTI-TN-5B1-4;Invitrogen) was tamed from a fetal bovine serum-containing medium to aserum-free medium (Ultimate Insect Serum-Free Medium; Invitrogen) over aperiod of about 1 month. Then, pIZT/V5-His-HBsAg was transformed intothe High Five strain tamed to the serum-free medium, using thegene-transfer lipid, Insectin-Plus (Invitrogen Co.). The transformantwas cultivated in a serum-free medium at 27° C. for 48 hours, and thenin a serum-free medium containing 400 μg/mL of the antibiotic zeocin(Invitrogen Co.) for 4 to 7 days, until the cells became confluent.

The supernatant was recovered by centrifugation at 1500×g, for 5minutes, and the HBsAg particles expressed were measured by Auszyme IIETA kit (Dainabot Co. Ltd.), to confirm the expression of HBsAgparticles.

Thus the resulting supernatant (1 L) was concentrated by ultrafiltration(the filter used was UK-200, ADVANTEC; exclusion molecular weight 200K)and then purified by an anion exchange column (DEAE-Toyopearl 650M; ToyoSoda), to obtain 2 mg of uniform HBsAg particles.

Example J Preparation of HBsAg Particles with Reduced Antigenicity

HBsAg particles can evoke anti-HBsAg antibodies in human wheninoculated. Therefore, modified HBsAg particles, in which theantigenicity of the major antigen S-protein was reduced, were prepared.

Specifically, a variant of HB virus isolated from a patient who wasinoculated with HB vaccine but developed hepatitis B (Carman W F,Zanetti A R, Karayiannis P, Waters J, Manzille G, Tanzi E, Zuckerman AJ, Thomas H G; Lancet Aug. 11, 1990; 336 (8711); 325-9; Chiou H L, Lee TS, Kuo J, Mau Y C, Ho M S, J Gen Virol 1997 October; 78 (Pt 10):2639-45) was introduced into the HBsAg particles.

In order to substitute the 145th Gly residue of the S-protein Bite ofthe plasmid pGLDLIIP39-RcT described in Example A to an Arg residue (themutation is indicated by an underline), two PCR primers coding5′-GCTGTACAAAACCTTCGGACAGAAACTGCACTTGTATTCC-3′ (SEQ ID NO: 19) and itscomplimentary sequence 5′-GGAATACAAGTGCAGTTTCTGTCCGAAGGTTTTGTACAGC-3′(SEQ ID NO: 20), were used to carry out PCR of the plasmidpGLDLIIP39-RcT using a QuickChange™ Site-Directed Mutagenesis Kit(Stratagene).

Specifically, as a thermostable DNA polymerase, Pfu DNA polymerase(Stratagene) was used, and the PCR schedule was: denaturation at 95° C.for 30 seconds; annealing at 55° C. for 1 minute; polymerizationreaction at 68° C. for 30 minutes; repetition of 30 cycles. Thereafter,the PCR product was treated with the restriction enzyme DpnI andtransformed into Escherichia coli DH5α, after which the vector DNA wasextracted from the colonies generated, and the mutated pGLDLIIP39-RcTplasmid was screened according to its nucleotide sequence. (Hereinafterreferred to as pGLDLIIP39-RcT (G145R)).

In the same manner as in Example A, the plasmid pGLDLIIP39-RcT (G145R)was transformed and cultivated in the synthetic medium High-Pi and8S5N-P400 to express HBsAg (G145R) particles.

From the recombinant yeast in the stationary growth phase (72 hoursafter starting incubation), crude cell extracts were prepared usingYeast Protein Extraction Reagent (Pierce Chemical Co.), separated bySDS-PAGE, and subjected to silver staining and Western blotting using ananti-S monoclonal antibody (Funakoshi Co.) to identify the HBsAg(G145R).

Thus, HBsAg (G145R) was found to be a protein with a molecular weight ofabout 52 kDa.

According to the method described in Example B, about 20 mg of thepurified HBsAg (G145R) particles were obtained from the above-describedcells (about 26 g) obtained from 2.5 L of the culture medium. TheS-antigenicity of HBsAg particles and HBsAg (G145R) particles wasmeasured using the Auszyme II EIA kit (Dainabot Co. Ltd.), which canonly detect the particle structure of HBsAg. The values were 1 for theformer and 0.2. for the latter.

In order to substitute the 129th Gln residue of the S-protein site ofthe above-described plasmid pGLDLIIP39-RcT (G145R) to an Arg residue(the mutation is indicated by an underline), two PCR primers coding5′-GCACGATTCCTGCTCGAGGAACCTCTATG-3′ (SEQ ID NO: 21) and itscomplimentary sequence 5′-CATAGAGGTTCCTCGAGCAGGAATCGTGC-3′ (SEQ ID NO:22), were used to carry out PCR for the plasmid pGLDLIIP39-RCT (G145R)using QuickChange™ Site-Directed Mutagenesis Kit (Stratagene).

Specifically, as a thermostable DNA polymerase, Pfu DNA polymerase(Stratagene) was used, and the PCR schedule was: denaturation at 95° C.for 30 seconds; annealing at 55° C. for 1 minute; polymerizationreaction at 68° C. for 30 minutes; repetition of 30 cycles.

Thereafter, the PCR product was treated with the restriction enzyme DpnIand transformed into Escherichia coli DH5α, after which the vector DNAwas extracted from the colonies generated, and the mutated plasmidpGLDLIIP39-RCT (G145R) (hereinafter referred to as pGLDLIIP39-RcT(Q129R, G145R)) was screened according to its nucleotide sequence.

In the same manner as in Example A, the plasmid pGLDLIIP39-RcT (Q129R,G145R) was transformed and cultivated in the synthetic medium High-Piand 8S5N-P400 to express HBsAg (Q129R, G145R) particles.

From the recombinant yeast in the stationary growth phase (72 hoursafter starting incubation), crude cell extracts were prepared usingYeast Protein Extraction Reagent (Pierce Chemical Co.), separated bySDS-PAGE, and subjected to silver staining and Western blotting using ananti-S monoclonal antibody (Funakoshi Co.) to identify the HBsAg (Q129R,G145R).

Thus, HBsAg (Q129R, C145R) was found to be a protein with a molecularweight of about 52 kDa.

According to the method described in Example B, about 20 mg of thepurified HBsAg (Q129R, G145R) particles were obtained from theabove-described cells (about 26 g) separated from 2.5 L of the culturemedium. The S-antigenicity of HBsAg particles and HBsAg (Q129R, G145R)particles was measured using Auszyme II EIA kit (Dainabot Co. Ltd.),which can only detect the particle structure of HBsAg. The values were 1for the former and less than 0.01 for the latter.

As described above, it is apparent that the HBsAg (Q129R, G145R)particles have low antigenicity and can be applied as a stable andeffective means for transporting substances, even in a living body.

INDUSTRIAL APPLICABILITY

As described above in detail, the present invention provides novelhollow nano particles, which may be used as a transporter for thespecific transportation and introduction of a substance into cells ortissues. Although the hollow nano particles hold a strong infectivitytowards particular cells or tissues, they are highly safe, since theycontain no viral genome, and may accordingly be widely applied in genetherapy or as DDS. Additionally, they are highly useful in industry,because they can be produced using a large-scale production system.

1. A transporter for transferring a substance into target cells or tissues, comprising a hollow nanoparticle obtained by expressing a hepatitis B virus surface antigen protein or mutant thereof capable of forming a particle in a eukaryotic cell, and incorporated therein is a substance to be introduced into the target cell or tissues, wherein the substance is selected from the group consisting of genes, oligonucleotides, natural or synthetic proteins peptides and drugs, wherein the substance is introduced into the transporter by simple diffusion, a charged lipid, ultrasonication or electroporation and wherein the expressed hepatitis B virus surface antigen or mutant thereof is the only hepatitis B virus-originated protein component of the hollow nanoparticle.
 2. The transporter according to claim 1, wherein the hepatitis B virus surface antigen protein is a hepatitis B virus surface antigen L-protein.
 3. The transporter according to claim 1, wherein the amino acid at position 129 of the S-protein site is substituted with arginine and the amino acid at position 145 of the S-protein is substituted with arginine. 